[13] Purification of the aa₃-Type Cytochrome-c Oxidase from Rhodopseudomonas Sphaeroides

This chapter describes the purification of the aa₃-type cytochrome-c oxidase from Rhodopseudomonas sphaeroides. The chapter outlines the procedures for the preparation of the bacterial membranes and purification of the oxidase. R. sphaeroides is a nonsulfur bacterium that, when grown anaerobically in the light, develops a photosynthetic apparatus. In the dark and in the presence of oxygen, the photosynthetic apparatus is suppressed and the bacterium utilizes a respiratory chain. The cytochrome-c oxidase from R. sphaeroides can easily and quickly be prepared in small amounts. The enzyme is immunologically related to that found both in yeast mitochondria and P. denitrificans. This oxidase appears to be structurally similar to other bacterial aa₃-type oxidases, though does not function as a proton pump. The success of the purification of the oxidase critically depends on the purity of the cytoplasmic membrane preparation.